In vitro and in vivo models for screening compounds to prevent glucocorticoid-induced bone destruction

ABSTRACT

The present invention demonstrates that glucocorticoid-induced bone disease is due to changes in the birth and death rate of bone cells using a murine model of glucocorticoid excess as well as bone biopsy specimens obtained from patients with glucocorticoid-induced osteoporosis. This invention demonstrates that glucocorticoid administration increases apoptosis of mature osteoblasts and osteocytes and decreases bone formation rate and bone mineral density accompanied by defective osteoblastogenesis and osteoclastogenesis in the bone marrow.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of U.S. provisional application60/105,805, filed Oct. 27, 1998, now abandoned.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to bone physiology. Morespecifically, the present invention relates to in vitro and in vivomodels for screening compounds to prevent glucocorticoid-induced bonedestruction.

[0004] 2. Description of the Related Art

[0005] The adverse effects of hypercortisolism on bone have beenrecognized for over 60 years (1), but the precise cellular and molecularbasis of these changes has remained elusive. Today, the iatrogenic formof the disease has become far more common than Cushing's syndrome andglucocorticoid-induced osteoporosis is now third in frequency followingpost-menopausal and senile osteoporosis (2).

[0006] Bone loss due to glucocorticoid excess is diffuse, affecting bothcortical and cancellous bone, but has a predilection for the axialskeleton. Spontaneous fractures of the vertebrae or ribs are, therefore,often presenting manifestations of the disorder (3,4). A cardinalfeature of glucocorticoid-induced osteoporosis is decreased boneformation (5). In addition, patients receiving long-term glucocorticoidtherapy sometimes develop collapse of the femoral head (osteonecrosis),but the mechanism underlying this is uncertain (6). Decreased boneformation, and in situ death of isolated segments of the proximal femursuggest that glucocorticoid excess may alter the birth and death of bonecells. Defective osteoblastogenesis has been reported to be linked toreduced bone formation and age-related osteopenia in the SAMP6 mouse(7). Besides the relationship between aberrant osteoblast production andosteoporosis, it has been recently shown that a significant proportionof osteoblasts undergo apoptosis (8), which raises the possibility thatthe premature or more frequent occurrence of osteoblast apoptosis couldcontribute to incomplete repair of resorption cavities and loss of bone.

[0007] Thus, the prior art is deficient in compounds that possess theadvantageous properties of glucocorticoids, namely anti-inflammatoryproperties, but do not cause bone loss or osteoporosis. The presentinvention provides for methods of screening compounds to fulfill thislong-standing need and desire in the art.

SUMMARY OF THE INVENTION

[0008] To demonstrate that glucocorticoid-induced bone disease is due tochanges in the birth or death rate of bone cells, a murine model ofglucocorticoid excess was used as well as bone biopsy specimens obtainedfrom patients with glucocorticoid-induced osteoporosis. This inventiondemonstrates that glucocorticoid administration decreases bone formationrate and bone mineral density accompanied by defectiveosteoblastogenesis and osteoclastogenesis in the bone marrow andincreases apoptosis of mature osteoblasts and osteocytes.

[0009] One object of the present invention is to provide methods toscreen compounds that retain the anti-inflammatory properties ofglucocorticoids yet do not result in bone loss or osteoporosis due toapoptosis of osteoblasts and osteocytes.

[0010] In one embodiment of the present invention, there is provided amethod of screening for compounds that reduce the bone deterioratingeffects of glucocorticoids, comprising the steps of: (a) contactingosteoblast and osteocyte cells with either a glucocorticoid alone or aglucocorticoid in combination with a test compound; and (b) comparingthe number of cells undergoing apoptosis following treatment with theglucocorticoid alone or following treatment with the glucocorticoid incombination with the test compound; wherein a lower number of apoptoticcells following treatment with the glucocorticoid in combination withthe test compound than with the glucocorticoid alone indicates that thetest compound reduces the bone deteriorating effects of theglucocorticoid. This embodiment also includes the aforementioned method,wherein the compound has little effect on the anti-inflammatoryproperties of the glucocorticoid, further comprising the step ofcomparing the anti-inflammatory response of the glucocorticoid incombination with the test compound to the anti-inflammatory response ofthe glucocorticoid alone; wherein essentially equivalentanti-inflammatory responses of the glucocorticoid alone and theglucocorticoid in combination with t h e test compound is indicates thatthe test compound both reduces the bone deteriorating effects, whileretaining the anti-inflammatory properties of the glucocorticoid;wherein said anti-inflammatory response is determined by models ofinflammation selected from the group consisting of the adjuvant-inducedarthritis model and hindlimb inflammation model.

[0011] In another embodiment of the present invention, there is provideda method of screening for glucocorticoid analogs that possess decreasedapoptotic properties towards osteoblast and osteocyte cells, comprisingthe steps of: (a) contacting the cells with either a glucocorticoid or aglucocorticoid analog; and (b) comparing the number of apoptotic cellsfollowing treatment with the glucocorticoid or the glucocorticoidanalog, wherein a lower number of apoptotic cells following treatmentwith the glucocorticoid analog than with the glucocorticoid indicatesthat the glucocorticoid analog possesses decreased apoptotic propertiestowards the cells. This embodiment also includes the aforementionedmethod, wherein the glucocorticoid analog retains anti-inflammatoryproperties, further comprising the step of: (c) comparing theanti-inflammatory response of the glucocorticoid in combination with atest compound to the anti-inflammatory response of the glucocorticoidalone, wherein essentially equivalent anti-inflammatory responses of theglucocorticoid alone and the glucocorticoid in combination with t h etest compound is indicative of a glucocorticoid analog that possessesdecreased apoptotic properties while retaining anti-inflammatoryproperties; wherein said anti-inflammatory response is determined bymodels of inflammation selected from the group consisting of theadjuvant-induced arthritis model and hindlimb inflammation model.

[0012] In yet another embodiment of the present invention, there isprovided a method of screening for compounds that stimulate bonedevelopment, comprising the steps of: (a) contacting osteoblast andosteocyte cells with either a glucocorticoid or a test compound; and (b)comparing the number of cells undergoing apoptosis following treatmentwith the glucocorticoid or the test compound; wherein a lower number ofapoptotic cells following treatment with the test compound than with theglucocorticoid indicates that the test compound stimulates bonedevelopment.

[0013] In still yet another embodiment of the present invention, thereis provided a method of screening for compounds that increase bonemineral density, comprising the steps of: (a) contacting osteoblast andosteocyte cells with either a glucocorticoid or a test compound; and (b)comparing the number of cells undergoing apoptosis following treatmentwith the glucocorticoid and the test compound; wherein a lower number ofapoptotic cells following treatment with the test compound than with theglucocorticoid is indicative of a compound that increases bone mineraldensity.

[0014] In the above-mentioned embodiments, contacting is selected fromthe group consisting of in vitro cell cultures and in vivo murine animalmodel and determination of apoptosis is selected from the groupconsisting of TUNEL, DNA fragmentation and immunohistochemical analysis.

[0015] Other and further aspects, features, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] So that the matter in which the above-recited features,advantages and objects of the invention, as well as others which willbecome clear, are attained and can be understood in detail, moreparticular descriptions of the invention briefly summarized above may behad by reference to certain embodiments thereof which are illustrated inthe appended drawings. These drawings form a part of the specification.It is to be noted, however, that the appended drawings illustratepreferred embodiments of the invention an d therefore are not to beconsidered limiting in their scope.

[0017]FIG. 1 shows photomicrographs of the effects of prednisolone onmurine vertebral cancellous bone. In panel A, is a longitudinal,panoramic section from a mouse receiving placebo and in panel B, asection from a mouse receiving prednisone. The histomorphometric readingarea is outlined. Toluidine blue stain, original magnification X25.

[0018]FIG. 2 shows quantification of CFU-OB and osteoclast progenitorsformed in ex vivo bone marrow cell cultures. Marrow cells were obtainedfrom the femurs of male mice after 27 d of exposure to placebo (whitebars) or 2.1 mg/kg/d of prednisolone (black bars). Cells from each mousewere cultured separately.

[0019]FIG. 3 shows the effect of prednisolone on murine osteoblastapoptosis. Osteoblasts were counted in undecalcified sections ofcancellous bone from the vertebral secondary spongiosa. In panel A, theplacebo group is shown and in panel B, the higher dose prednisolonegroup. Apoptotic cells in this experiment were identified using. TUNELand morphometric features such as nuclear fragmentation and condensationof chromatin (arrows). Methyl green counterstain viewed with Nomarskidifferential interference microscopy, original magnification X400.

[0020]FIG. 4 shows the effect of prednisolone on murine osteocyteapoptosis. The cells were counted in undecalcified sections of femoralmetaphyseal cortical bone. In panel A, the placebo group is shown and inpanel B, the higher dose prednisolone group. Apoptotic osteocytes(arrowheads) are seen in close proximity to normal cells. Methyl greencounterstain viewed with Nomarski differential interference microscopy,original magnification X630.

[0021]FIG. 5 shows the effect of chronic prednisone treatment onapoptosis in human bone. TUNEL-positive osteoblasts (arrowheads) andosteocytes (arrows) were absent from normal subjects (FIG. 5A) but wereclearly identified in patients with prednisone-induced osteoporosis(FIG. 5B and FIG. 5C). Approximately 5% of the osteocytes and 30% of theosteoblasts were apoptotic. The photomicrographs are from transiliacbone biopsy specimens. Methyl green counterstain viewed with Nomarskidifferential interference microscopy, original magnification X630.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Glucocorticoid-induced bone disease is characterized b ydecreased bone formation and in situ death of isolated segments of bone(osteonecrosis) suggesting that glucocorticoid excess, the third mostcommon cause of osteoporosis, may affect the birth or death rate of bonecells thus reducing their numbers. To examine this, prednisolone wasadministered to 7-month-old mice for 27 days and decreased bone density,serum osteocalcin and cancellous bone area along with trabecularnarrowing were found. These changes were accompanied by diminished boneformation and turnover, as determined by histomorphometric analysis oftetracycline-labeled vertebrae, and impaired osteoblastogenesis andosteoclastogenesis, as determined by ex vivo bone marrow cell cultures.In addition, the mice exhibited a 3-fold increase in osteoblastapoptosis in vertebrae and showed apoptosis in 28% of the osteocytes inmetaphyseal cortical bone. As in mice, an increase in osteoblast andosteocyte apoptosis was documented in patients withglucocorticoid-induced osteoporosis. Decreased production of osteoclastsexplains the reduction in bone turnover while decreased production andapoptosis of osteoblasts would account for the decline in bone formationand trabecular width. Furthermore, accumulation of apoptotic osteocytesmay contribute to osteonecrosis. These findings provide evidence thatglucocorticoid-induced bone disease arises from changes in the numbersof bone cells.

[0023] The present invention is directed towards methods of screeningcompounds that retain the anti-inflammatory properties ofglucocorticoids while lacking the bone degeneration propertiesassociated with long-term administration due to apoptosis of osteoblastsand osteocytes.

[0024] The present invention is further directed towards methods ofscreening compounds that promote bone regeneration b y inhibiting theapoptosis of osteoblasts and osteocytes.

[0025] As used herein, the terms “glucocorticoid” and “glucocorticoidanalog” is defined as substances that bind to the glucocorticoidreceptor.

[0026] As used herein, the term “apoptosis” refers to programmed celldeath with nuclear fragmentation and cell shrinkage as detected bymorphological criteria and Terminal Uridine Deoxynucleotidal TransferaseNick End Labeling (TUNEL) staining.

[0027] As used herein, the terms “anti-inflammatory response” or“anti-inflammatory property” refers to preventing the induction ofcytokines and other events that lead to T cell activation. Severalmodels of inflammation are routinely used in the art, including theadjuvant-induced arthritis model and hindlimb inflammation model whichare well known to those having ordinary skill in this art (54, 55).

[0028] As used herein, the term “bone mineral density” refers to bonemass as defined by Dual-Energy X-Ray Absorbtiometry (DEXA).

[0029] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion:

EXAMPLE 1

[0030] Animals

[0031] Male Swiss Webster mice (Charles River Laboratories, Stone Ridge,N.Y.) were electronically tagged (Biomedic Data System Inc., Maywood,N.J.) and kept in plastic cages (3-5 animals per cage) under standardlaboratory conditions with a 12 hr dark, 12 hr light cycle and aconstant temperature of 20° C. and humidity of 48%. All mice were fed ona standard rodent diet (Agway RMH 3000, Arlington Heights, Ill.)containing 22% protein, 5% fat, 5% fiber, 6% ash, 3.5 Kcal/g, 1.0 IUvitamin D3/g, 0.97% calcium and 0.85% phosphorus with water ad libitum.The animals and food supply were weighed at one week intervalsthroughout the experiment. Studies were approved by the UAMS Division ofLaboratory and Animal Medicine.

EXAMPLE 2

[0032] Glucocorticoid Administration—Experimental Design

[0033] Bone mineral density (BMD) determinations were done a t two weekintervals to identify the peak adult bone mass of the mice, which wasreached between 5 and 6 months-of-age (9). Animals at peak bone masswere used to avoid obscuring the negative impact of glucocorticoidexcess on bone mineral density by the confounding effects of increasedlinear and radial growth. Before the experiment began, bone mineraldensity measurements were repeated to allocate the animals into groups(n=4−5) with equivalent spinal density values. The mice (7-mo-old)received placebo or prednisolone, a synthetic glucocorticoid analog thatdoes not require hepatic hydroxylation and has minimal mineralocorticoidactivity, thus eliminating the need for potassium supplementation orsodium restriction (10,11). Implantation of pellets releasing 0.5mg/kg/d of prednisolone (the no effect dose) did not decrease bonemineral density. Therefore, two doses were used, 0.7 mg/kg/d (lowerdose) and 2.1 mg/kg/d (higher dose), chosen from pilot studies tobracket the dose (1.4 mg/kg/d) that invariably causes densitometricevidence of bone loss. These doses were administered for 27 days bysubcutaneous implantation of slow-release pellets (Innovative Researchof America, Sarasota, Fla.). Bone mineral density measurements wereobtained at the beginning of the experiment and 27 dayspost-implantation. For dynamic histomorphometric measurements,tetracycline HCl (30 mg/kg body weight) was given intraperitoneally 17and 23 days post-implantation. After 27 days, the mice were sacrificed,serum and urine specimens were taken, bone marrow aspirates wereobtained from the right femur for ex vivo marrow cell cultures and theleft femur and lumbar vertebrae were prepared for histomorphometricanalysis. Livers were examined for fatty infiltration as a sign ofprednisolone toxicity. The weight of the seminal vesicles (mg/100 g bodyweight) was used as an index of the androgen status of the animals (12).To help interpret these measurements, a separate group of animals wasorchidectomized (n=5).

EXAMPLE 3

[0034] Bone densitometry

[0035] Dual-energy X-ray absorptiometry (DEXA) was used to determineglobal (whole body minus the head), spinal and hindquarters bone mineraldensity in live mice (7,9). The scans done at 27 days after pelletimplantation were analyzed using the ‘Compare’ technique, in which theevaluation is based on the exact positioning and region of interestplacement of the baseline scan. Accuracy of the DEXA measurements wasdemonstrated by the strong linear relationship between ash weight andbone mineral content at each region (7). Over the 18 months, thecoefficient of variation for the bone mineral density of aplastic-embedded whole mouse skeleton was 3.0% (n=146).

EXAMPLE 4

[0036] Serum and Urine Biochemical Measurements

[0037] Serum osteocalcin was measured by radioimmunoassay using a goatanti-murine osteocalcin and murine osteocalcin as tracer and standard(Biomedical Technologies, Stoughton, Mass.). Urinary freedeoxypyridinoline excretion was determined by a microtiter competitiveenzyme immunoassay (Pyrilinks-D, Metra Biosystems, Mountain View,Calif.) and was expressed as a ratio to the urinary creatinine.

EXAMPLE 5

[0038] Bone Histomorphometric Analysis

[0039] The distal femora and lumbar vertebrae were fixed in 4° C.Millonig's phosphate-buffered 10% formalin, pH 7.4, embeddedundecalcified in methyl methacrylate and stained (7,9,13). Thehistomorphometric examination was done with a computer and digitizertablet (OsteoMetrics Inc. Version 3.00, Atlanta, Ga.) interfaced to aZeiss Axioscope (Carl Zeiss, Inc., Thornwood, N.Y.) with a drawing tubeattachment. All cancellous measurements were two-dimensional, confinedto the secondary spongiosa and made at X400 magnification (numericalaperture 0.75). The terminology and units used are those recommended bythe Histomorphometry Nomenclature Committee of the American Society forBone and Mineral Research (14). The trabecular width and osteoid widthwere measured directly. Trabecular spacing and number were calculated(15). Only TRAPase-positive cells were included in the osteoclastperimeter. The rate of bone formation (μm²/μm/d) and turnover (%/d) werecalculated (7).

EXAMPLE 6

[0040] Detection and Quantification of Osteoblasts and Osteoclasts in ExVivo Bone Marrow Cultures

[0041] One femur from each mouse was flushed with 5 ml of phenolred-free αMEM (Gibco BRL, Gaithersburg, Md.) containing 10% FBS(Hyclone, Logan, Utah) to obtain marrow cells. After the cells wererinsed and resuspended to obtain a single cell suspension, the nucleatedcell count was determined using a Coulter Counter. Cells from eachanimal were cultured separately.

[0042] The number of colony-forming unit-fibroblast (CFU-F) andCFU-osteoblast (CFU-OB) present in the bone marrow preparations weredetermined (16-18). Briefly, cells were seeded at 1.5×10⁶ per 10 cm²well for the determination of CFU-F number and maintained for 10 days inphenol red-free αMEM containing 15% preselected FBS, 50 μM ascorbic acidand 10 mM β-glycerophosphate (Sigma Chemical Co, St. Louis, Mo.) withone-half of the medium replaced after 5 days. After fixation in neutralbuffered formalin and staining with hematoxylin, colonies containing aminimum of 20 fibroblastoid cells were enumerated. Cells were seeded at2.5×10⁶ cells per 10 cm² well for the determination of CFU-OB number andcultured for 25-28 days as described above for CFU-F. After fixation in50% ethanol and 18% formaldehyde, cultures were stained using VonKossa's method to visualize and enumerate colonies containingmineralized bone matrix.

[0043] Osteoclast formation in bone marrow cultures was assessed inreplicate cultures (4-6 from each animal) maintained for 9 days in thepresence of αMEM, 10% FBS and 10 nM 1.25(OH)₂D₃ (7). Briefly, marrowcells were cultured at 1.5×10⁶ per 2 cm² well on 13 mm round Thermanoxdisks and maintained for 8 days in the presence of 10% FBS in αMEMsupplemented with 10⁻⁸ M 1.25(OH)₂D₃ (provided by Dr. Milan Uskokovic,Hoffman-LaRoche, Nutley, N.J.). At the end of the experiment, cells wereprocessed for the autoradiographic detection of bound ¹²⁵I-calcitonin(¹²⁵I-CT) and stained for tartrate-resistant acid phosphatase. Becausemany osteoclasts in murine bone possess only one nucleus (7), it isimpossible to distinguish between preosteoclasts and mononuclearosteoclasts in ex vivo cultures of murine bone marrow cells. Therefore,mononucleated and multinucleated cells that both bind ¹²⁵I-CT andexpress TRAPase were designated as osteoclastic cells. The number ofosteoclasts formed in this assay is a reflection of the number ofosteoclast progenitors present in the bone marrow aspirate and thenumber of stromal/osteoblastic support cells that form during theculture period.

[0044] The number of CFU-F colonies, CFU-OB colonies, and osteoclasticcells formed from the marrow cells of each animal was expressed as thenumber per femur, which was calculated b y multiplying the number ofcolonies or osteoclasts obtained per 10⁶ cells seeded at the initiationof the cultures by the total number of marrow cells obtained from theanimal.

EXAMPLE 7

[0045] Measurement of Apoptosis in Undecalcified Bone Sections

[0046] Sections were mounted on silane-coated glass slides (ScientificDevice Lab, Inc., Des Plains, Ill.), deplasticized and incubated in 10mM citrate buffer, pH 7.6, in a microwave oven at 98° C. for 5 minutes.Slides were then incubated with 0.5% pepsin for 30 minutes at 37° C.Apoptotic cells were detected by the TUNEL reaction(transferase-mediated biotin-dUTP nick end-labeling) using Klenowterminal deoxynucleotidyl transferase (Oncor, Gaithersburg, Md.) insections counterstained with 1% methyl green. The TUNEL reaction wasnoted within cell nuclei and the cells whose nuclei were clearly brownfrom the peroxidase-labeled anti-digoxigenin antibody instead of theblue-green from the methyl green were interpreted as positive.Plastic-embedded sections of weaned rat mammary tissue were used as apositive control. Negative controls were made b y omitting thetransferase. Morphological changes characteristic of apoptosis wereexamined carefully to minimize ambiguity regarding the interpretation ofresults. With these precautions, TUNEL has been unequivocally associatedwith apoptosis (19). In addition, TUNEL has been used with DNAfragmentation and immunohistochemical studies to demonstrate apoptosisof osteoblastic cells and osteoblasts both in vitro and in vivo (8,20).Apoptosis was also assessed in transiliac bone biopsy specimens takenfrom two patients with glucocorticoid-induced osteoporosis (22- and36-yr-old, receiving 15 to 25 mg/d of prednisone for 3 to 6 yr) and from12 age-, sex- and race-matched controls (13). Two longitudinal sectionswere examined from each patient and control subject. Osteoblasts wereidentified as cuboidal cells lining the osteoid-covered trabecularperimeter (7,9,13). Osteocytes were identified inside lacunae inmineralized bone.

EXAMPLE 8

[0047] Statistics

[0048] Differences in the bone densitometry values were determined usingthe percentage change in BMD from baseline. Dose response relations weretested by one-way ANOVA. To further evaluate changes in bonehistomorphometry, a Student's t test was used to assess for significantdifferences between group means, after testing for equivalence ofvariances and normal distribution of data. The significance of therelative frequency of apoptotic cells was determined with the χ²statistic. P values less than 0.05 were considered significant (21).

EXAMPLE 9

[0049] Demonstration of Bone Loss in Mice Receiving Prednisolone

[0050] In mice implanted with the higher dose of prednisolone, globaland spinal BMD at 27 days were significantly lower than those found inthe mice that were implanted with placebo pellets (TABLE I). Thedecrease in global bone mineral density was dose dependent (P<0.05).Demonstrating the expected propensity for the axial skeleton,glucocorticoid-induced loss of bone mineral density was less conspicuousat the hindquarters. The levels of serum osteocalcin, a marker ofosteoblast activity, were decreased more than 50% when compared toplacebo, while urinary deoxypyridinoline excretion was not significantlydifferent between the groups (TABLE I). These effects were not due tochanges in food intake, body weight or androgen status (TABLE II). Inaddition, hepatic fatty infiltration was absent. TABLE I Bone MineralDensity (BMD) and Serum and Urine Biochemical Measurements inPrednisolone-treated Mice Measurement Placebo 0.7 mg/kg/d 2.1 mg/kg/dGlobal BMD (% change) −2.7 ± 2.1  −5.0 ± 2.2* −6.6 ± 1.9† Spinal BMD (%change) −3.1 ± 3.0  −6.8 ± 3.2  −8.7 ± 3.5* Hindquarters BMD  0.4 ± 10.4−3.8 ± 8.0  −3.4 ± 6.9  (% change) Osteocalcin (μg/L) 93.8 ± 11.5  63.0± 27.7*  46.4 ± 13.8† Deoxypyridinoline 78.3 ± 9.3  63.6 ± 14.7 81.5 ±11.3 (μM/mM creatinine)

[0051] TABLE II Food Intake, Body Weight and Seminal Vesicle Weight inPrednisolone-treated Mice Measurement Placebo 0.7 mg/kg/d 2.1 mg/kg/dFood Intake (g/d) 3.4 ± 0.6 3.6 ± 0.2  3.7 ± 0.4 Body Weight (g) 37.9 ±6.0  33.8 ± 4.3  32.2 ± 4.2 Seminal Vesicle Weight 74.6 ± 14.6 92.7 ±8.7  83.1 ± 6.9 (mg/100 g body weight)

EXAMPLE 10

[0052] Effects of Glucocorticoid Administration on Vertebral BoneHistomorphometry

[0053] Consistent with the bone mineral density results, in the animalsreceiving the higher dose, there was a 40% decline in the vertebralcancellous bone area and a 23% decline in trabecular width (P<0.01)(TABLE III). In both prednisolone groups, there was a trend towardsincreased trabecular spacing and there was decreased trabecular numberin the lower dose group indicating that some trabecular profiles wereentirely resorbed.

[0054] In the higher dose group, osteoid area decreased by 29%, osteoidperimeter by 34% and osteoid width by 27% (P<0.01). A trend towarddecreased osteoblast and osteoclast perimeters was found in the animalsreceiving the higher dose. There was, however, a 3-fold increase in theempty erosion cavities (devoid of osteoclasts) or reversal perimeter.The tetracycline-based histomorphometry showed that prednisoloneadministration caused a 26% decrease in the mineralizing perimeter(P<0.05). In addition, a dose-dependent decrease in the mineralappositional rate was noted (P<0.05); this decline was 22% with thelower dose and 40% with the higher dose. Furthermore, there was a 53%decrease in the rate of bone formation with the higher dose (P<0.01),which correlated with the vertebral cancellous bone area (r=0.57,P<0.05), indicating that the glucocorticoid-induced decreases in bonearea were associated with a reduction in the rate of bone formation.Bone turnover, expressed as a percentage of the bone area per day, alsodecreased in a dose-dependent manner (P<0.05). TABLE III VertebralCancellous Bone Histomorphometry in Swiss Webster Mice After 27 Days ofPrednisolone Administration Histomorphometric Determination Placebo 0.7mg/kg/d 2.1 mg/kg/d Bone area/Tissue area(%) 10.4 ± 1.4  6.9 ± 2.1  6.3± 1.7† Trabecular width (μm) 48.0 ± 2.4  48.6 ± 4.3  37.1 ± 4.4†Trabecular spacing (μm) 423 ± 69  712 ± 302 546 ± 125 Trabecular number(per mm) 1.66 ± 0.6  1.44 ± 0.47 1.77 ± 0.33 Osteoid area/Bone area (%)2.1 ± 0.2 2.2 ± 0.8  1.5 ± 0.2† Osteoid perimeter/Bone perimeter 15.1 ±2.1  15.8 ± 5.1   9.9 ± 1.1† (%) Osteoid width (μm) 2.6 ± 0.4 2.0 ± 0.3 1.9 ± 0.3* Osteoblast perimeter/Bone perimeter 1.2 ± 0.9 2.2 ± 0.2 0.5± 0.4 (%) Osteoclast perimeter/Bone perimeter 2.7 ± 1.1 2.6 ± 0.5 1.1 ±1.7 (%) Reversal perimeter/Bone perimeter 2.5 ± 2.3 3.2 ± 2.2  7.2 ±1.1† Mineralizing perimeter/Bone perimeter 12.9 ± 0.5  13.9 ± 5.6   9.5± 2.5* (%) Mineral appositional rate 1.23 ± 0.11  0.96 ± 0.11*  0.74 ±0.20† (μm/d) Bone formation rate/Bone perimeter 0.15 ± 0.02 0.13 ± 0.04 0.07 ± 0.03† (μm2/(m/d) Bone turnover (%/d) 0.68 ± 0.09  0.46 ± 0.12* 0.24 ± 0.11†

EXAMPLE 11

[0055] Effects of Glucocorticoid Administration on Osteoblastogenesisand Osteoclastogenesis

[0056] In bone marrow cell cultures from the animals receiving thehigher dose, there was no significant change in CFU-F colonies (1250±374vs. 698±104, NS). However, the number of CFU-OB colonies decreased by86% (375±257 SD vs. 54±14, P<0.05) and the number of osteoclastic cellsformed in response to 1.25(OH)₂D₃ in ex vivo marrow cultures decreasedby 65% (1387±920 vs. 492±311, P<0.05) (FIG. 2).

EXAMPLE 12

[0057] Effects of Glucocorticoid Administration on Apoptosis

[0058] Counting a total of 973 osteoblasts, there was a 3-fold increasein osteoblast apoptosis in the vertebral cancellous bone of micereceiving the higher dose of prednisolone when compared to controls(2.03%±0.34 vs. 0.66%±0.07, P<0.05). Morphological changes typical ofapoptosis accompanied the TUNEL-positive osteoblasts and includedsharply defined, condensed chromatin plastered against the nuclearmembrane, nuclear fragmentation and cell shrinkage (FIGS. 3A and 3B).

[0059] In addition, prednisolone caused the appearance of apoptoticosteocytes in cortical bone sections taken from femora (FIGS. 4A and4B). Whereas none of the osteocytes exhibited apoptotic features in thecontrol animals, 28% of 131 cortical osteocytes were apoptotic in theanimals receiving the higher dose. Osteocyte apoptosis was restricted tosmall groups of cells in the center of the femoral metaphyseal cortexand were absent from vertebral cortical bone. The apoptotic osteocyteswere identified in close proximity to normal osteocytes, in contrast tothe large homogenous areas of dead and dying cells typical of cellnecrosis. An increase in apoptotic hypertrophic chondrocytes and bonemarrow cells was also noted in mice receiving either dose ofprednisolone. Osteoclast apoptosis was not observed.

EXAMPLE 13

[0060] Demonstration of Apoptotic Osteoblasts and Osteocytes in Patientswith Glucocorticoid-Induced Osteoporosis

[0061] In transiliac bone biopsies taken from two patients,TUNEL-positive osteoblasts and osteocytes were clearly identified inboth (FIGS. 5B and 5C) but were absent from specimens taken from 12age-, sex- and race-matched controls (FIG. 5A). As in the murine model,bone histomorphometry from these two patients showed the changesexpected with chronic glucocorticoid therapy (5): reduced cancellousbone area (11.1 and 8.8%, normal is 22.4±1.2 SEM), decreased trabecularwidth (62 and 118 μm, normal is 161±9), decreased osteoblast perimeter(2.1 and 2.3%, normal is 7.6±0.4), decreased osteoclast perimeter (0 and0.4%, normal is 0.9±0.2), increased reversal perimeter (13.5 and 15.4%,normal is 6.9±0.7) and diminished bone formation rate (0.02 and 0.05μm²/μm/d, normal is 0.095±0.012). In the cancellous bone of thesespecimens, approximately 5% of the osteocytes and 30% of the osteoblastswere apoptotic. Apoptosis of osteoclasts or cortical osteocytes was notobserved. A transiliac bone biopsy represents a much smaller sample ofthe human skeleton than the murine femur and lumbar vertebrae representof the mouse skeleton. Therefore, it is not surprising that thepercentage of apoptotic osteoblasts and osteoclasts was different in thehuman and murine specimens.

EXAMPLE 14

[0062] Early Effects on Bone Resorption

[0063] To directly establish whether glucocorticoids initiallyaccelerate bone resorption in the mouse, the vertebral cancellous bonehistology were examined in an additional group of somewhat younger mice(5-mo-old) after 7 days administration of the higher dose ofprednisolone or placebo (n=5). It was found that whereas prednisolonecaused a 59% decrease in the osteoblast perimeter (5.2%±1.5 SD vs.2.1±1.1, P<0.005), the osteoclast perimeter increased 96% (0.51% ±0.34vs. 1.00±0.41, P<0.05).

SUMMARY

[0064] The choice of the mouse for these studies was based on itsvalidity as a model of the bone loss associated with loss of sexsteroids (9,22) and with senescence (7), but the mouse also has severaladvantages over other animals (TABLE IV). In the mouse, glucocorticoidadministration consistently induces axial, greater than appendicular,bone loss without weight loss or hypogonadism, accompanied byhistological indices of impaired osteoblast function, thus reproducingthe major features of the human disease (2-5). Although the doses usedin the studies described herein were higher in relation to body weightthan in humans, they were only mildly higher than the dose determined byserial bone densitometry to have no effect and were consistent with themuch higher metabolic clearance of glucocorticoids and other compoundsin laboratory animals than in humans (35-37). Nonetheless, thesimilarity of the glucocorticoid-induced increases in apoptotic cellsand bone histomorphometric features in mice and humans indicates thatthe observations in the mouse are not due to pharmacologicaldifferences.

[0065] The effects of glucocorticoids were examined after 27 days, aperiod equivalent in the mouse to about 3 to 4 years in humans. Thus,these findings represent long-term, rather than acute effects. Althougha significant correlation was found between the severity of thecancellous bone loss and the extent of reduction in bone formation,several other lines of evidence imply that some of the observed boneloss was due to an early increase in bone resorption which had subsidedby the time of examination. First, there was suggestive evidence ofcomplete loss of some trabeculae (TABLE III). Second, based on the boneturnover measured in the placebo group which must be close to the ratefound in all the animals at the beginning of the study, even with totalsuppression of bone formation, the initial rate of bone turnover couldhave accounted only for an exponential decline in cancellous bone areaof 18%, whereas a 40% decrease was observed. Finally, an early increasein osteoclast perimeter was confirmed b y histomorphometric examinationof vertebral cancellous bone after 7 days of prednisoloneadministration.

[0066] By 27 days of prednisolone administration, bone resorption fellto, or below, normal, as indicated by the downward trend in theosteoclast perimeter, normal urinary deoxypyridinoline excretion andprofound decrease in osteoclastogenesis. The persistent increase inerosion cavities devoid of osteoclasts, measured as the reversalperimeter, merely indicates delayed bone formation (38), and has beenpreviously observed in glucocorticoid-treated patients (5,39).Consequently, the present invention emphasizes the relevant findings at27 days to chronic, rather than short-term, glucocorticoidadministration to humans.

[0067] Vertebral cancellous bone in adult mice undergoes sequential,coupled bone remodeling that is qualitatively similar to that occurringin human bone (7,9). Many of the changes in cellular, osteoid andtetracycline-based histological indices induced b y glucocorticoidadministration can be accounted for by a reduction in the activationfrequency of bone remodeling, the main determinant of the rate of boneturnover (40), which is an inevitable consequence of the substantialdecrease in osteoclastogenesis that was observed. Although a reductionin bone turnover will not by itself cause bone loss, the decrease intrabecular width, which was the major structural change observed, isusually the result of incomplete cavity repair. This is, at least inpart, due to inadequate osteoblast recruitment, either from diminishedproduction or ineffective migration to the bone surface (40). Thereduction in osteoblastogenesis was of sufficient magnitude to explainthe decrease in bone formation rate, and would also have contributed tothe inadequate osteoblast recruitment and consequent decline intrabecular width. Thus, the inhibitory effect of glucocorticoids onearly bone cell progenitors in the bone marrow can account for many ofthe in vivo observations.

[0068] The data herein also bear on recent ideas concerning therelationships between early osteoblast and osteoclast progenitors in thebone marrow. Although mature osteoclasts and osteoblasts are neededsuccessively at each bone surface site that is being remodeled, thesecells are needed simultaneously as the basic multicellular unit (whichis the instrument of bone remodeling) progresses through or across thesurface of bone (41). The necessary parallel production of executivecells is accomplished by signals that originate from early members ofthe stromal cell-osteoblast family, which support in various ways theproduction of mononuclear preosteoclasts in the bone marrow (42). Thedemonstration herein of a marked reduction in the numbers of both CFU-OBand osteoclast progenitors derived from ex vivo bone marrow cellcultures makes it likely that glucocorticoid administration inhibits theproliferation and/or differentiation of the stromal cell-osteoblastfamily at a n early stage, leading to a reduction in the number ofmature, matrix-secreting osteoblasts as well as the osteoblastic cellsthat support osteoclast development. A direct inhibitory effect ofglucocorticoids on osteoclast precursor proliferation is not excluded bythe data herein, but would be less easy to reconcile with the finding ofa n early increase in the osteoclast perimeter.

[0069] Some osteoblasts become osteocytes and some become lining cells,but these fates combined do not account for all t h e osteoblastsinitially present. Although migration along or away from the bonesurface is possible, death has always seemed the most likely alternativefate (43). Osteoblasts in remodeling bone undergo apoptosis with afrequency sufficient to account for most or all of those missing (8).Based on the dynamic histomorphometry at the murine vertebral secondaryspongiosa and a wall width of about 15 μm (7,9,14), the mean active lifespan of an osteoblast was calculated on cancellous bone by dividing wallwidth by the mineral appositional rate. From this calculation, the meanactive lifespan of a murine osteoblast is about 12 days or 288 hours.The prevalence of osteoblast apoptosis in the present study was 0.0066in the placebo group. The following relationship was applied:

t _(Ap)/288=0.0066/f _(Ap),

[0070] where t_(Ap) is the mean duration (in hours) of the DNAfragmentation phase of apoptosis that is detected by TUNEL, and f_(Ap)is the fraction of osteoblasts that undergoes apoptosis and based on avalue of t_(Ap) of about 3 hours, determined previously for regeneratingliver (44), the corresponding value for f_(Ap) in the placebo group is0.6. Thus, the low prevalence of apoptosis in the placebo group isconsistent with studies of human bone that 50-70% of osteoblasts undergoapoptosis, and that only a minority become osteocytes or lining cells(43).

[0071] In the animals receiving the higher dose of prednisolone, theprevalence of apoptosis was 0.0203. With prednisolone administration,phagocytosis of the apoptotic cells would b e suppressed and it wasestimated that t_(Ap) could be doubled (45). Wall width was reduced toabout 8 μm and mineral appositional rate to 0.74 μm/d, so that theactive lifespan of an osteoblast is about 260 hours. In thesecircumstances, the corresponding value for f_(Ap) in the prednisolonegroup is 0.9. Although there is some uncertainty to the assumptions usedfor these estimates, the approach does help explain the data anddisclose the devastating impact of glucocorticoid excess on osteoblastsurvival. The higher proportion of osteoblasts showing features ofapoptosis in glucocorticoid-treated mice and human subjects couldindicate no more than prolongation of the time needed for completion ofthe process, but it is more likely that glucocorticoids induceapoptosis, either prematurely in cells already destined for this fate orin cells otherwise destined to become lining cells or osteocytes. Ineither case, the mean active lifespan of osteoblasts would be shortenedand less bone formed. Thus, t h e reduction in bone formation byglucocorticoids could be due to increased death as well as decreasedbirth of osteoblasts.

[0072] Osteocytes are long-lived but not immortal cells. In human ribcortical bone, their lifespan has been estimated at about 20 years (47);if bone remains unremodeled for a longer time, the osteocytes die, asrevealed by empty lacunae and hypermineralized perilacunar bone,referred to as micropetrosis (48). Osteocyte death in cancellous bone,indicated by absence of lactic dehydrogenase activity, increases inprevalence with age in the upper femur but not in the vertebrae (49),probably because of the higher bone turnover in the spine. Empty lacunaeand enzyme absence can reveal the fact, but not the mode, of death.Osteocyte apoptosis has recently been detected in human iliac cancellousbone and its prevalence was increased by pharmacological induction ofestrogen deficiency (19). The present invention demonstrated thatchronic glucocorticoid administration, both to mice and to humanpatients, likewise increases the prevalence of osteocyte apoptosis. Theproportion of apoptotic osteocytes was much higher than of osteoblasts,reflecting the unique unavailability of osteocytes for phagocytosisbecause of their anatomic isolation from scavenger cells, and the needfor extensive degradation to small molecules to dispose of the cellsthrough the narrow canaliculi. As a result, the process is prolonged andaffected cells accumulate.

[0073] The network of osteocytes probably participates in the detectionof microdamage and the transmission of signals that lead to its repairby remodeling (50). Disruption of the network b y osteocyte apoptosiscould compromise this mechanism, leading to microdamage accumulation andincreased bone fragility (51). Second, chronic glucocorticoidadministration sometimes leads to so-called aseptic or avascularnecrosis of bone (6). Glucocorticoid-induced osteocyte apoptosis, acumulative and unrepairable defect, would explain the correlationbetween total dose and incidence of avascular necrosis of bone (53) andits occurrence after glucocorticoid administration had ceased.

[0074] In conclusion, the present invention has demonstrated that themouse is a valid and informative model of glucocorticoid-induced bonedisease, not confounded by weight loss or sex-steroid deficiency, andthat many of the effects of chronic glucocorticoid administration onbone can be explained by decreased birth of osteoblast and osteoclastprecursors and increased apoptosis of mature osteoblasts and osteocytes.TABLE IV Confounding Factors with Glucocorticoid Administration AnimalsFactors Rats (23, 24) Paradoxical increase in cancellous bone mass*,decreased food intake and weight. Rabbits and Inconsistent changes inbone density and cancellous dogs (25-27) bone area, weight loss, hepaticfatty infiltration. Ewes (28-30) Histological changes resembleglucocorticoid-treated patients but corresponding changes in bonedensity and cancellous bone area are inconsistent.

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[0131] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

[0132] One skilled in the art will appreciate readily that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those objects, ends and advantagesinherent herein. The present examples, along with the methods,procedures, treatments, molecules, and specific compounds describedherein are presently representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention asdefined by the scope of the claims.

1. A method of screening for compounds that reduce the bonedeteriorating effects of glucocorticoids, comprising the steps of: (a)contacting osteoblast and osteocyte cells with either a glucocorticoidalone or said glucocorticoid in combination with a test compound; and(b) comparing the number of osteoblast and osteocyte cells undergoingapoptosis following treatment with said glucocorticoid alone orfollowing treatment with said glucocorticoid in combination with saidtest compound, wherein a lower number of apoptotic cells followingtreatment with said glucocorticoid in combination with said testcompound than with said glucocorticoid alone indicates that the testcompound reduces the bond deteriorating effects of said glucocorticoid.2. The method of claim 1, wherein said contacting is selected from thegroup consisting of in vitro cell cultures and in vivo murine animalmodel. 3 (canceled)
 4. The method of claim 1, wherein said test compoundhas little effect on the anti-inflammatory properties of saidglucocorticoid, further comprising the step of: (c) comparing theanti-inflammatory response of said glucocorticoid in combination withsaid test compound to the anti-inflammatory response of saidglucocorticoid alone, wherein essentially equivalent anti-inflammatoryresponses of said glucocorticoid alone and said glucocorticoid incombination with said test compound indictes that the test compound bothreduces the bone deteriorating effects while retaining theanti-inflammatory properties of said glucocorticoid.
 5. The method ofclaim 4, wherein said contacting is in an in vivo murine animal model.6. The method of claim 4, wherein said anti-inflammatory response isdetermined by models of inflammation selected from the group consistingof the adjuvant-induced arthritis model and hindlimb inflammation model.7. A method of screening for glucocorticoid analogs that possessdecreased apoptotic properties towards osteoblast and osteocyte cells,comprising the steps of: (a) contacting said cells with either aglucocorticoid or a glucocorticoid analog; and (b) comparing the numberof apoptotic cells following treatment with said glucocorticoid or saidglucocorticoid analog, wherein a lower number of apoptotic cellsfollowing treatment with said glucocorticoid analog than with saidglucocorticoid is indicative of a glucocorticoid analog that possessdecreased apoptotic properties towards said cells.
 8. The method ofclaim 7, wherein said contacting is selected from the group consistingof in vitro cell cultures and in vivo murine animal model. 9 (canceled)10. The method of claim 7, wherein said glucocorticoid analog retainsanti-inflammatory properties, further comprising the step of: comparingthe anti-inflammatory response of said glucocorticoid in combinationwith a test compound to the anti-inflammatory response of saidglucocorticoid alone, wherein essentially equivalent anti-inflammatoryresponse of said glucocorticoid alone and said glucocorticoid incombination with said test compound indicates that the glucocorticoidanalog possesses decreased apoptotic properties while retaininganti-inflammatory properties.
 11. The method of claim 10, wherein saidcontacting is in an in vivo murine animal model.
 12. The method of claim11, wherein said anti-inflammatory response is determined by models ofinflammation selected from the group consisting of the adjuvant-inducedarthritis model and hindlimb inflammation model.
 13. A method ofscreening for glucocorticoid analogs that stimulate bone development,comprising the steps of: (a) contacting osteoblast and osteocyte cellswith either a glucocorticoid or a test compound; and (b) comparing thenumber of said cells undergoing apoptosis following treatment with saidglucocorticoid and said test compound, wherein a lower number ofapoptotic cells following treatment with said test compound than withsaid glucocorticoid is indicative of a compound that stimulates bonedevelopment.
 14. The method of claim 13, wherein said contacting isselected from the group consisting of in vitro cell cultures and in vivomurine animal model.
 15. The method of claim 13, wherein determinationof said apoptosis is slected from the group consisting of TUNEL, DNAfragmentation and immunohistochemical analysis.
 16. A method ofscreening for compounds that increase bone mineral density, comprisingthe steps of: (a) contacting osteoblast and osteocyte cells with eithera glucocorticoid or a test compound; and (b) comparing the number ofsaid cells undergoing apoptosis following treatment with saidglucocorticoid and said test compound, wherein a lower number ofapoptotic cells following treatment with said test compound than withsaid glucocorticoid is indicative of a compound that stimulates bonedevelopment.
 17. The method of claim 16, wherein said contacting isselected from the group consisting of in vitro cell cultures and in vivomurine animal model.
 18. The method of claim 16, wherein determinationof said apoptosis is selected from the group consisting of TUNEL, DNAfragmentation and immunohistochemical analysis.